Figure 6

MagD stability and cleavage are affected in mutants lacking MagB and MagE. (A) Crude extracts of P. aeruginosa PAO1 (WT) or knock-out strains lacking indicated proteins were run on SDS-PAGE and immune-developed with polyclonal anti-MagD antibodies. The Δop mutant¹⁵ that lacks the entire mag operon was used as control. In the wild-type background MagD appears as 165 kDa native and 100 kDa cleaved forms. The 100 kDa form is absent in ΔmagB and ΔmagC backgrounds. An additional higher molecular weight form observed in ΔmagE could correspond to unprocessed MagD still containing the signal peptide (*). (B) Crude extracts of indicated strains lacking MagB (ΔmagB) or expressing magB or magD in trans were analyzed as in (A). (C) Bacteria were further fractionated into membranes and periplasm showing that MagB governs macroglobulin membrane localization and cleavage to the 100 kDa form. Note the presence of 165 kDa form of MagD exclusively in the periplasm of the magB mutant. This form is subject to rapid degradation as visualized by products of smaller sizes in the ΔmagB::magD strain. In the ΔmagB::magB strain, membrane localization was restored and MagD appears as 165 kDa and 100 kDa forms, similarly to wild-type. EFTu, DsbA and TagQ antibodies were used as loading controls for whole bacterial extracts, periplasm and membrane samples, respectively. Gels have had lanes selected/cropped for clarity.